Lipoxygenase

ABSTRACT

The inventors have found a novel fungal lipoxygenase from  Magnaporthe salvinii  and determined its sequence. They have sequenced the gene and cloned it into  E. coli  and deposited the clone. Oligonucleotide probes based on the sequence information are useful for screening a eukaryotic library to obtain a lipoxygenase. The lipoxygenase is useful in baking and in a detergent.

FIELD OF THE INVENTION

The present invention relates to a lipoxygenase and a polynucleotideencoding it.

BACKGROUND OF THE INVENTION

Lipoxygenase (EC 1.13.11.12) is an enzyme that catalyzes the oxygenabonof polyunsaturated fatty acids such as linoleic acid, linolenic acid andarachidonic acid, which contain a cis,cis-1,4-pentadiene unit andproduces hydroperoxides of these fatty acids. The enzyme is widelydistributed in plants and animals. A number of lipoxygenase genes havebeen isolated from various plant and mammalian sources.

On the other hand, only a limited number of microbial lipoxygenases areknown, and no lipoxygenase gene of microbial origin has been described.Su and Oliw, J. Biological Chemistry, 273 (21), 13072-79 (1998) describea lipoxygenase from Gaeumannomyces graminis.

SUMMARY OF THE INVENTION

The inventors have found a novel fungal lipoxygenase and determined itssequence, which can be used for the production of the enzyme inindustrial scale. They have cloned the gene into E. coli and depositedthe clone.

Accordingly, the invention provides a lipoxygenase which is:

a) a polypeptide encoded by a DNA sequence cloned into plasmid pUC19present in Escherichia coli deposited as DSM 14139,

b) a polypeptide having an amino acid sequence as the mature peptideshown in SEQ ID NO: 1, or which can be obtained therefrom bysubstitution, deletion, and/or insertion of one or more amino acids,

c) an analogue of the polypeptide defined in (a) or (b) which:

i) has at least 50% homology with said polypeptide,

ii) is immunologically reactive with an antibody raised against saidpolypeptide in purified form,

iii) is an allelic variant of said polypeptide, or

d) a polypeptide encoded by DNA that hybridizes under low stringencyconditions with a complementary strand of

i) the DNA sequence cloned into plasmid pUC19 present in Escherichiacoli deposited as DSM 14139 or

ii) the DNA sequence of SEQ ID NO: 1 encoding the mature polypeptide ora subsequence thereof having at least 100 nucleotides.

The invention also provides a polynucleotide which comprises:

a) the partial DNA sequence encoding a mature lipoxygenase cloned into aplasmid present in Escherichia coli DSM 14139,

b) the partial DNA sequence encoding a mature lipoxygenase shown in SEQID NO: 1,

c) an analogue of the sequence defined in a) or b) which encodes alipoxygenase and

i) has at least 60% homology with said DNA sequence, or

ii) hybridizes at high stringency with a complementary strand of saidDNA sequence or a subsequence thereof having at least 100 nucleotides,

iii) is an allelic variant thereof, or

d) a complementary strand of a), b) or c).

Other aspects of the invention provide a nucleic acid construct and arecombinant expression vector comprising the polynucleotide, arecombinant host cell comprising the construct or the vector, and amethod of producing a lipoxygenase by cultivating the cell. Further, theinvention provides a method of screening a eukaryotic library to obtaina lipoxygenase and an oligonucleotides probe useful for screening.Finally, the invention provides use of the lipoxygenase in baking and ina detergent.

DETAILED DESCRIPTION OF THE INVENTION

Genomic DNA Source

A lipoxygenase gene of the invention may be derived from a filamentousfungus, e.g. an Ascomycota, particularly Magnaporthaceae, such as astrain of Magnaporthe, particularly Magnaporthe salvinii Cattaneo(Mycologia 64 (1), 110 (1972)). The species is also known under thesynonyms Curvularia sigmoidea, Helminthosporium sigmoideum,Leptosphaeria salvinii Nakataea sigmoidea, Sclerotium oryzae andVakrabeeja sigmoidea. An example is the strain M. salvinii IFO 6642.

Alternatively, the gene may be isolated from Pyriculadia, e.g. P. oryzaeor P. grisea, e.g. P. oryzae IFO 30517. The IFO strains are available oncommercial terms from Institute for Fermentation, Osaka (IFO), 17-85,Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan.

The lipoxygenase gene may be isolated from these organisms using probesdesigned on the basis of the DNA sequences in this specification.

A strain of Escherichia coli containing a lipoxygenase gene from M.salvinii IFO 6642 was deposited by the inventors under the terms of theBudapest Treaty with the DSMZ—Deutsche Sammmlung von Microorganismen undZelikulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE, Germany.The deposit date was 28 Feb. 2001, and the accession number was DSM14139.

Production of Lipoxygenase by Cultivation of Transformant

The lipoxygenase of the invention may be produced by transforming asuitable host cell with a DNA sequence encoding the lipoxygenase,cultivating the transformed organism under conditions permitting theproduction of the enzyme, and recovering the enzyme from the culture.

The host organism may be a eukaryotic cell, in particular a fungal cell,such as a yeast cell or a filamentous fungal cell, e.g. a strain ofAspergillus, Fusarium, Trichoderma or Saccharomyces, particularly A.niger, A. oryzae, F. graminearum, F. sambucinum, F. cerealis or S.cerevisiae. The production of the lipoxygenase in such host organismsmay be done by the general methods described in EP 238,023 (NovoNordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).

Properties of LOX

The lipoxygenase of the invention is able to oxidize a wide range ofsubstrates containing a cis-cis-pentadienyl moiety. Thus, it acts onpolyunsaturated fatty acids such as linoleic acid (18 carbon atoms, 2double bonds), linolenic acid (18:3), arachidonic acid (20:4),eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6).It also acts on substrates other than fatty acids, such as methyllinoleate and probably also triglycerides. The enzyme has a very lowMichaelis constant (K_(M)) for linoleic acid and a high specificity(V_(max)/K_(M)) towards this substrate.

The lipoxygenase from M. salvinii is a 9-lipoxygenase, i.e. it oxidizesthe double bond between carbon atoms 9 and 10 in linoleic acid andlinolenic acid.

The lipoxygenase from M. salvinii has optimum activity around pH 7, andit is highly active over a broad pH range 3-12, having more than 50% ofoptimum activity in the range pH 6-11. It is stable after overnightincubation at pH 5-11.

The native lipoxygenase from M. salvinii has optimum activity at 50-60°C. It is quite active at 40-60° C., and the activity begins to declineat 70° C. The lipoxygenase is stable after 30 minutes incubation at pH 7at temperatures up to 50° C.

The reaction rate for recombinant lipoxygenase (expressed in A. oryzae)increases nearly ten times at the optimal temperature for catalysiscompared to the rate obtained at room temperature. The maximum reactionrate is obtained at 67.5° C. A steep decrease in rate constant is seenabove the temperature optimum. It is believed that glycosylation rendersthe recombinant enzyme more stable towards heat than the wild-typeenzyme.

The recombinant lipoxygenase is quite stable at temperatures up to 50°C. for at least one hour. The activity drops in a linear fashion athigher temperatures between 50-60° C., and no activity is detected afterincubations above 60° C. for one hour. No activity loss is detectedduring incubation at temperatures below 45° C.

Frozen solutions of the lipoxygenase lose some activity during storage.With addition of 10% glycerol there is no discernible activity lossafter two weeks storage at −20° C., and the enzyme survived repeatedcycles of thaw-freeze without loss of activity.

The lipoxygenase of the invention has good stability in the presence ofanionic surfactants. Thus, the lipoxygenase from M. salvinii is stablein the presence of 400 ppm of LAS (linear alkyl-benzene sulfonate).

Use of Lipoxygenase

The lipoxygenase can be used for green flavor synthesis, e.g. nonenalfrom 9-hydoperoxide of linolenic acid. The synthesis may be done inanalogy with Whitehead et al.1995, Cereal foods world 40(4),193-197 andU.S. Pat. No. 4,769,243.

The lipoxygenase can also be used for plant hormone synthesis asdescribed in JP H11-29410.

Also the lipoxygenase is a good oxidant of carotenoids, so it can beused for bleaching of foodstuffs such as flour, oil or marine foodincluding carotenoids or carotenoid-like pigments.

The oxidation activity can be utilized for cross-linking of protein,oil, starch, fiber and mixture of these. Cross-linking of chemicalcompounds can be utilized for synthesis of polymer to give plastic fiberor plastic resin. It can be used for bleaching as a detergent forphenolic, carotenoid or fatty stains or dinginess. Or it can be used forbleaching of waste water or textile dye.

Lipoxygenase can be used for bleaching of plant or marine food materialscontaining of carotenoids. Thus it could be used for bleaching of flourfor bread, noodle or pasta, or bleaching of fish meat or fish oilcontaining astaxanthin.

It also can be used for cross-linking of protein, oil, starch,plant-fiber or mixture of these in presence of fatty acid, oil or fats.It is useful to change the texture or physical properties of foodstuffor to control of flavor for fat and oil, or to produce polymers made ofnatural staff beside food use. Cross-linked compounds can be chemicalcompounds, e.g. phenolic, carbonyl, carboxyl or amide compounds ormixture of these. It could be used for synthesis of plastic fiber orresin.

Other usages of lipoxygenase can be the synthesis of flavor compoundsuch as hexanal or hexenal together as synergy effect of hydroperoxidelyase. Or in case plant material is used as the source of above twoenzymes, lipoxygenase can be added to it to improve the yield of flavorcompound. The similar can be done for synthesis of plant or animalhormones.

Finally it can be used as bleaching agent. It can be used in detergentsfor bleaching of phenolic, carotenoid, fatty stains or dinginess ofclothes. Or it can be used for bleaching of textile dye or dye for pulpindustry in waste water or changing of dye texture.

Recombinant Expression Vector

The expression vector of the invention typically includes controlsequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a selectable marker, atranscription terminator, a repressor gene or various activator genes.The vector may be an autonomously replicating vector, or it may beintegrated into the host cell genome.

Production by Cultivation of Transformant

The lipoxygenase of the invention may be produced by transforming asuitable host cell with a DNA sequence encoding the lipoxygenase,cultivating the transformed organism under conditions permitting theproduction of the enzyme, and recovering the enzyme from the culture.

The host organism may be a eukaryotic cell, in particular a fungal cell,such as a yeast cell or a filamentous fungal cell, e.g. a strain ofAspergillus, Fusarium, Trichoderma or Saccharomyces, particularly A.niger, A. oryzae, F. graminearum, F. sambucinum, F. cerealis or S.cerevisiae. The production of the lipoxygenase in such host organismsmay be done by the general methods described in EP 238,023 (NovoNordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).

The enzyme can be purified in one step by cation-exchange chromatographyto homogeneity.

Nucleotide Probe

A nucleotide probe may be designed on the basis of the DNA sequence ofSEQ ID NO: 1 or the polypeptide sequence of SEQ ID NO: 2, particularlythe mature peptide part. The probe may be used in screening forLOX-encoding DNA as described below.

A synthetic oligonucleotide primer may be prepared by standardtechniques (e.g. as described in Sambrook J, Fritsch E F, Maniatis T(1989) Molecular cloning: a laboratory manual (2^(nd) edn.) Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.) on the basis of the maturepart of the amino acid sequence in SEQ ID NO: 2 or the correspondingpart of the DNA sequence. It may be a degenerate probe and willtypically contain at least 20 nucleotides.

Screening of Eukaryotic DNA Library

A polypeptide with lipoxygenase activity may be obtained by a methodcomprising:

a) preparing a eukaryotic DNA library,

b) screening the library to select DNA molecules which hybridize to theprobe described above,

c) transforming host cells with the selected DNA molecules,

d) cultivating the transformed host cells to express polypeptidesencoded by the DNA molecules, and

e) assaying the expressed polypeptides to select polypeptides havinglipoxygenase activity.

The eukaryotic DNA library may be prepared by conventional methods. Itmay include genomic DNA or double-stranded cDNA derived from suitablesources such as those described above.

Molecular screening for DNA sequences may be carried out by polymerasechain reaction (PCR) followed by hybridization.

In accordance with well-known procedures, the PCR fragment generated inthe molecular screening may be isolated and subcloned into a suitablevector. The PCR fragment may be used for screening DNA libraries by e.g.colony or plaque hybridization.

Hybridization

The hybridization is used to indicate that a given DNA sequence isanalogous to a nucleotide probe corresponding to a DNA sequence of theinvention. The hybridization may be done at low, medium or highstringency. One example of hybridization conditions is described indetail below.

Suitable conditions for determining hybridization between a nucleotideprobe and a homologous DNA or RNA sequence involves presoaking of thefilter containing the DNA fragments or RNA in 5×SSC (standard salinecitrate) for 10 min, and prehybridization of the filter in a solution of5×SSC (Sambrook et al. 1989), 5× Denhardt's solution (Sambrook et al.1989), 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA(Sambrook et al. 1989), followed by hybridization in the same solutioncontaining a random-primed (Feinberg, A. P. and Vogelstein, B. (1983)Anal. Biochem. 132:6-13), ³²P-dCTP-labeled (specific activity >1×10⁹cpm/μg) probe for 12 hours at approx. 45° C. The filter is then washedtwo times for 30 minutes in 2×SSC, 0.5% SDS at a temperature of at least55° C., particularly at least 60° C., more particularly at least 65° C.,e.g. at least 70° C., or at least 75° C.

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using an x-ray film.

Alignment and Homology

The lipoxygenase and the nucleotide sequence of the invention may havehomologies to the disclosed sequences of at least 75% or at least 85%,particularly at least 90% or at least 95%, e.g. at least 98%.

For purposes of the present invention, alignments of sequences andcalculation of homology scores were done using a Needleman-Wunschalignment (i.e. global alignment), useful for both protein and DNAalignments. The default scoring matrices BLOSUM50 and the identitymatrix are used for protein and DNA alignments respectively. The penaltyfor the first residue in a gap is −12 for proteins and −16 for DNA,while the penalty for additional residues in a gap is −2 for proteinsand −4 for DNA. Alignment is from the FASTA package version v20u6 (W. R.Pearson and D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA”, Methods inEnzymology, 183:63-98).

EXAMPLES

Materials and Methods

Molecular cloning techniques are described in Sambrook et al. (1989).

The following commercial plasmids and E. coli strains were used forsub-cloning and DNA library construction:

pT7Blue (Novagen)

pUC19 (TOYOBO, Japan)

E. coli JM109 (TOYOBO, Japan)

E. coli DH12S (GIBCO BRL, Life Technologies, USA)

Labeling and detection of hybridization probe was done usingDIG-labeling and detection Kit (Boehringer Manheim). Nylon membraneHybond-N+ (Amersham, England) was used for DNA transfer for colonyhybridization.

Soybean lipoxygenase (type I-B) (cat.# L7315) and astaxanthin (cat.#A-9335) was supplied by Sigma. b-carotene (cat.# 031-05533) weresupplied by Wako.

Media and Buffer Solution

COVE-ar: per liter 342.3 g sucrose, 20 ml COVE salt solution, 10 mMacryl amide, 15 mM CsCl₂, 30 g Agar noble (Difco)

COVE2-ar: per liter 30 g sucrose, 20 ml COVE salt solution, 10 mMacrylamide, 30 g Agar noble (Difco)

COVE salt solution: per liter 26 g KCl, 26 g MgSO₄-7H₂O, 76 g KH₂PO₄, 50ml Cove trace metals.

Cove trace metals: per liter 0.04 g NaB₄O₇—10H₂O, 0.4 g CuSO₄—5H₂O, 1.2g FeSO₄—7H₂O, 0.7 g MnSO₄—H₂O, 0.7 g Na₂MoO₂—2H₂O, 0.7 g ZnSO₄—7H₂O.

AMG trace metals: per liter 14.3 g ZnSO₄—7H₂O, 2.5 g CuSO₄—5H₂O, 0.5 gNiCl₂, 13.8 g FeSO₄, 8.5 g MnSO₄, 3.0 g citric acid.

YPG: per liter 4 g yeast extract, 1 g KH₂PO₄, 0.5 g MgSO₄—7H₂O, 15 gglucose, pH 6.0.

STC: 0.8 M Sorbitol, 25 mM Tris pH 8, 25 mM CaCl₂.

STPC: 40% PEG4000 in STC buffer.

Cove top agarose: per liter 342.3 g sucrose, 20 ml COVE salt solution,10 mM Acetamide, 10 g low melt agarose.

MS-9: per liter 30 g soybean powder, 20 g glycerol, pH 6.0.

MDU-2Bp: per liter 45 g maltose-1H₂O, 7 g yeast extract, 12 g KH₂PO₄, 1g MgSO₄—7H₂O, 2 g K₂SO₄, 5 g Urea, 1 g NaCl, 0.5 ml AMG trace metalsolution pH 5.0.

Host Organism

Aspergillus oryzae BECh2 is described in WO 00/39322. It is a mutant ofJaL228 (described in WO98/123000), which is a mutant of IFO4177.

Transformation of A. oryzae

Aspergillus oryzae strain BECh2 was inoculated in 100 ml of YPG mediumand incubated at 32° C. for 16 hours with stirring at 80 rpm. Grownmycelia was collected by filtration followed by washing with 0.6 M KCland re-suspended in 30 ml of 0.6 M KCl containing Glucanex® (Novozymes)at the concentration of 30 μl/ml. The mixture was incubated at 32° C.with the agitation at 60 rpm until protoplasts were formed. Afterfiltration to remove the remained mycelia, protoplasts were collected bycentrifugation and washed with STC buffer twice. The protoplasts werecounted with a hematitometer and re-suspended in a solution ofSTC:STPC:DMSO (8:2:0.1) to a final concentration of 1.2×10⁷protoplasts/ml. About 4 μg of DNA was added to 100 μl of protoplastsolution, mixed gently and incubated on ice for 30 minutes. 1 μl STPCbuffer was added to the mixture and incubated at 37° C. for another 30minutes. After the addition of 10 ml of Cove top agarose pre-warmed at50° C., the reaction mixture was poured onto COVE-ar agar plates. Theplates were incubated at 32° C. for 5 days.

SDS-PAGE

SDS polyacrylamide electrophoresis was carried out using thecommercialized gel PAGEL AE6000 NPU-7.5L (7.5T%) with the apparatusAE-6400 (Atto, Japan) following the provided protocol. 15 μl of samplewas suspended in 15 μl of 2× conc. of sample loading buffer (100 mMTris-HCl (pH 6.8), 200 mM Dithiothreitol, 4% SDS, 0.2% Bromophenol blueand 20% glycerol) and boiled for 5 minutes. 20 μl, of sample solutionwas applied to a polyacrylamide gel, and subjected for electrophoresisin the running buffer (25 mM Tris, 0.1% SDS, 192 mM Glycine) at 20 mAper gel. Resulting gel was stained with SYPRO Orange and detected bymolecular Imager FX (BIO-RAD).

Assays for Lipoxygenase Activity

Spectrophotometric Assay

Lipoxygenase activity was determined spectrophotometrically at 25° C. byfollowing the formation of hydroperoxides with the absorbance at 234 nm.To 0.98 ml of the buffer (50 mM KH₂PO₄/NaHPO₄, pH 7.0), 10 μl ofsubstrate solution (10 mM linolenic acid dispersed with 0.2% Tween20)was added and the reaction was started by the addition of 10 μl ofenzyme solution. One unit causes an increase in absorbance at 234 nm of0.001/min.

FOX Assay

The assay was initiated by the addition of 20 μl enzyme solution to 80μl of 50 mM each buffer containing 0.7 mM linolenic acid dispersed with0.02% of Tween 20 using Hiscotron, and incubated for 10 min. The assaywas terminated by the addition of 900 μl of FOX reagent: sulfuric acid(25 mM), xylenol orange(100 μM), iron(II) sulfate (100 μM), butylatedhydroxytoluen (4 mM) in methanol:water (9:1). Blanks contained onlysubstrate solution during the incubation, but enzyme solution was addedafter the addition of FOX reagent. The yellow color of acidified xylenolorange was converted to a blue color by the lipid hydroperoxide-mediatedoxidation of Fe²⁺ ions with the dye. Absorbance of the Fe³⁺ complex at620 nm was measured 1 hour after the addition of FOX reagent.

Bleaching Assay

Bleaching effect by lipoxygenase was examined spectrophotometrically at25° C. by following the absorbance at 470 nm. The pigment solution wasprepared as follows. 150 ul of stock pigment solution (1 mg each pigmentin I ml chloroform) was evaporated to be dry. Then 30 ml of the buffer(50 mM KH₂PO₄NaHPO₄, pH 7.0) with 0.3% of Tween 20 was added slowly andthe pigment was dissolved. To 0.98 ml of the pigment solution, 10 μl ofsubstrate solution (10 mM linolenic acid dispersed with 0.2% of Tween20)was added and the reaction was started by the addition of 10 μl ofenzyme solution.

Example 1 Cloning of Genomic LOX Gene from M. salvinii

Genomic DNA from Magnaporthe salvinii was digested with Sac I andseparated on 1.0% agarose gel. Around 2.5 kbp of DNA digestion wasrecovered from the gel and ligated with BAP treated pUC19 linearized bySac I. Ligation mixture was transformed into E. coli DH12S to constructa partial genomic library. It was screened, and a lipoxygenase-positiveE. coli colony was isolated and the plasmid, termed pSG28, wasrecovered. The plasmid pSG28 contained a 2.5 kbp Sacl genomic fragmentthat contained the presumed LOX homologue sequence. The sequence of 1973bp out of 2.5 kbp is shown as SEQ. ID 1.

Introns were identified and are indicated in SEQ ID NO: 1. The splicesites were predicted as described in S. M. Hebsgaard et al., NucleicAcids Research, 1996, Vol. 24, No. 17, 3439-3452.

The presumed open reading frame consisted of 1851 bp, and the deducedamino acid sequence corresponded to 617 amino acids, shown as SEQ ID NO:2. The molecular mass was estimated as 67500 Da.

The E. coli DH12S harboring plasmid pSG28 was deposited at DSMZ as DSM14139 with the accession date 2001-02-28.

Example 2 Expression of M. salvinii LOX in A. oryzae

Construction of Expression Plasmid

The partial genomic sequence of M. salvinii genomic gene was amplifiedby PCR using pSG28 as a template. Primer 3 and 4 (SEQ ID NO: 3 and 4)were designed to make BamH I and Xho I sites at both ends of the PCRproduct (nucleotides 4-9 of primer 3 and 5-10 of primer 4,respectively). PCR reaction mixture comprised of 2.5 mM dNTP, 30 pmoleach of primer 3 and 4, 5 units of LA taq polymerase (Takara) andsupplied GC buffer 1. Reaction condition was shown below. LA taqpolymerase was added to the reaction mixture after step 1. StepTemperature Time 1 98° C. 10 mins 2 96° C. 20 sec 3 55° C. 45 sec 4 72°C. 30 sec 5 72° C. 10 mins* Step 2 to Step 4 were repeated 30 times.

PCR amplified 1.9 kb fragment was isolated and cloned into pT7Blueresulting in pSG29.

The plasmid pSG29 was digested by BamHII and XhoI and 1.9 kb of fragmentwhich contained the LOX gene was ligated with pMT2188 digested withBamHI and XhoI. The plasmid pMT2188 has a modified Aspergillus nigerneutral amylase promoter, Aspergillus nidulans TPI leader sequence,Aspergillus niger glucoamylase terminator, Aspergillus nidulans amdSgene as a marker for fungal transformation and S. cerevisiae ura3 as themarker for E. coli transformation. Transformation was done with E. coliDB6507 in which pyrF gene is deficient and can be complemented with S.cerevisiae Ura3. Resulting plasmid was termed pSG30.

a Expression of M. salvinii LOX in A. oryzae

A. oryzae BECh2 was transformed with the plasmid pSG30 and selectionpositive transformants were isolated. Transformants were grown on COVE2-ar at 32° C. for 5 days and inoculated to 100 ml of MS-9 shakingflask. After the cultivation with vigorous agitation at 32° C. for 1day, 3 ml of each culture was transferred to 100 ml of MDU-2Bp inshaking flask to cultivate at 32° C. for 3 days. Culture broth wascentrifuged at 3500 rpm for 10 minutes and supernatant was collected.

Lipoxygenase activities of the supernatant were determinedspectrophotometrically as described before. Positive transformantsshowed about 100,000 U/ml culture broth while untransformed A. oryzaeBECh2 showed no activity. Culture supernatant was also subjected toSDS-PAGE analysis. Positive transformants showed 80-100 kDa smear bandwhich indicated the protein was heavily glycosylated. Untransformed A.oryzae BECh2 did not show any significant bands.

Example 3 Substrate Specificity of Lipoxygenase

Kinetic parameters for a number of substrates were determined bystandard methods for the M. salvinii lipoxygenase. V_(max) K_(M)V_(max)/K_(M) Substrate (μmol/min/mg) (μM) (μmol/min/mg/μM) Linoleicacid 2.63 1 2.557 Na linoleate 2.07 0.41 5.061 Linoelaidic acid Noactivity No activity No activity Linolenic acid 1.9 0.4 4.488Eicosadienoic acid 2.02 11 0.177 Arachidonic acid 2.44 5.5 0.446Linoleoyl chloride 0.97 12 0.080 Methyl linoleate 0.82 30 0.026Linoleoyl acetate 0.77 9 0.085 Linoleoyl alcohol 1.4 8 0.175

For comparison, one substrate was also tested with soybean lipoxygenase.V_(max) K_(M) V_(max)/K_(M) Substrate (μmol/min/mg) (μM)(μmol/min/mg/μM) Linoleic acid 12.3 230 0.054

Example 3 pH Dependence of Lipoxygenase Activity

The relative activity of the M. salvinii lipoxygenase at various pHvalues was determined by the FOX assay described above, using thefollowing buffers: 50 mM citric acid/sodium citrate (pH 2.21-3.73) ,KH₂PO₄Na₂HPO₄ (pH 5.30,6.17), Tris/HCl (pH 7.01,8.02), glycylglycineNaCl/NaOH (pH 9.33-11.0). pH Relative Activity (%) 2.21 7.11 2.90 20.63.73 27.7 5.30 60.0 6.17 83.7 7.01 100 8.02 92.9 9.33 82.6 11.0 77.7

Example 4 Temperature Dependence of Lipoxygenase Activity

The effect of temperature on the M. salvinii lipoxygenase was studied by10 min incubation at pH 7.0. Temperature Relative Activity (%) 25 50.140 90.0 50 100 60 99.6 70 60.4

Example 5 Bleaching Effect of Lipoxygenases

The bleaching effect of M. salvinii LOX was examined. Soybean L1 wasincluded for comparison. β-carotene and astaxanthin were used aspigments. Time (min) M. salvinii Soybean L1 β-carotene 0 0.3783 0.35750.4 0.3791 0.3616 0.8 0.3729 0.3601 1.2 0.3702 0.362 1.4 0.3685 0.36021.8 0.3651 0.3602 2.2 0.3633 0.3595 2.6 0.3486 0.3595 3 0.341 0.3594ΔA470/min 0.0121 0.00005 LOX activity 2.652 1.962 Astaxanthin 0 0.52920.5026 0.4 0.5244 0.5029 0.8 0.5177 0.505 1 0.5166 0.5025 1.4 0.5120.5013 1.8 0.5004 0.4993 2.2 0.4876 0.4985 2.6 0.4714 0.4986 3 0.45660.498 ΔA470/min 0.0239 0.0021 LOX activity 2.4952 2.018

The results show the M. salvinii LOX bleaches the pigment solutions.Soybean LOX showed little effect on bleaching.

1. An isolated lipoxygenase which is: a) a polypeptide encoded by a DNAsequence cloned into plasmid pUC19 present in Escherichia coli depositedas DSM 14139, b) a polypeptide having an amino acid sequence as themature peptide shown in SEQ ID NO: 1, or which can be obtained therefromby substitution, deletion, and/or insertion of one or more amino acids,c) an analogue of the polypeptide defined in (a) or (b) which: i) has atleast 50% homology with said polypeptide, ii) is immunologicallyreactive with an antibody raised against said polypeptide in purifiedform, iii) is an allelic variant of said polypeptide, or d) apolypeptide encoded by DNA that hybridizes under low stringencyconditions with a complementary strand of i) the DNA sequence clonedinto plasmid pUC19 present in Escherichia coli deposited as DSM 14139 orii) the DNA sequence of SEQ ID NO: 1 encoding the mature polypeptide ora subsequence thereof having at least 100 nucleotides.
 2. Thelipoxygenase of claim 1 which is derived from a filamentous fungus, e.g.an Ascomycota, such as a strain of Magnaporthe, particularly M.salvinii, more particularly strain IFO
 6642. 3. Isolated DNA comprisinga nucleic acid sequence which encodes the lipoxygenase of claim
 1. 4. Anisolated polynucleotide which comprises: a) the partial DNA sequenceencoding a mature lipoxygenase cloned into a plasmid present inEscherichia coli DSM 14139, b) the partial DNA sequence encoding amature lipoxygenase shown in SEQ ID NO: 1, c) an analogue of thesequence defined in a) or b) which encodes a lipoxygenase and i) has atleast 60% homology with said DNA sequence, or ii) hybridizes at highstringency with a complementary strand of said DNA sequence or asubsequence thereof having at least 100 nucleotides, iii) is an allelicvariant thereof, or d) a complementary strand of a), b) or c).
 5. Anucleic acid construct comprising the nucleic acid sequence of claim 3operably linked to one or more control sequences capable of directingthe expression of the lipoxygenase in a suitable expression host.
 6. Arecombinant expression vector comprising the nucleic acid construct ofclaim 5, a promoter, and transcriptional and translational stop signals.7. A recombinant host cell comprising the nucleic acid construct ofclaim
 5. 8. A method for producing a lipoxygenase comprising cultivatingthe host cell of claim 7 under conditions conducive to production of thelipoxygenase, and recovering the lipoxygenase.
 9. An oligonucleotideprobe which consists of at least 20 nucleotides and which encodes apartial polypeptide sequence of SEQ ID NO:
 2. 10. A method for obtaininga polypeptide with lipoxygenase activity, comprising: a) preparing aeukaryotic DNA library, b) screening the library to select DNA moleculeswhich hybridize to the probe of claim 8, c) transforming host cells withthe selected DNA molecules, d) cultivating the transformed host cells toexpress polypeptides encoded by the DNA molecules, and e) assaying theexpressed polypeptides to select polypeptides having lipoxygenaseactivity.
 11. A method for preparing a dough or a baked product madefrom dough, comprising adding the lipoxygenase of claim 1 to the dough.12. A dough composition comprising the lipoxygenase of claim
 1. 13. Adetergent composition comprising a surfactant and the lipoxygenase ofclaim
 1. 14. The detergent composition of the preceding claim whereinthe surfactant is anionic.
 15. A process for oxidizing a polyunsaturatedfatty acid comprising contacting the acid with the lipoxygenase of claim1 in the presence of air.
 16. (canceled)